Browsing by Author "Gunathilaka, P.A.D.H.N."

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    DNA Fingerprinting of Thunnus obesus and Thunnus albacores Fish Species for Proper Identification in Large Scale Fish Processing Industry
    (Uva Wellassa University of Sri Lanka, 2016) Perera, D.R.C.; Gunathilaka, P.A.D.H.N.; Rodrigo, W.W.P.; Athapaththu, A.M.M.H.; Bulumulla, P.B.A.I.K.
    Detection of species substitution has become an important topic within the food industry and there is a growing need for rapid, reliable, and reproducible tests to verify species in commercial fish and seafood products. The effects of species substitution are far-reaching and include economic fraud, health hazards, and illegal trade of protected species. In Sri Lanka tuna fish industry is a rapid developing field. However, the species identification prior to the processing is achieved through morphological characteristics, which is not a reliable method. Therefore, the aim of this study was to develop a diagnostic method by combining Polymerase Chain Reaction with Restriction digestion to differentiate Thunnus obesus (bigeye tuna) and Thunnus albacores (yellowfin tuna) species in order to facilitate the fish processing industries and fish exporters by developing the test for species confirmation. Deoxy ribonucleic acid (DNA) extracted from muscle tissues of T obesus and T albacores were analyzed. DNA was amplified using primers flanking a region of cytochrome b gene of 558 by and digested using two restriction endonucleases, EcoNI and Scat A product having band sizes of 187 by and 371 by was observed from T albacores after digesting with EcoNI. The digestive product by Scal resulted 215 by and 343 by band sizes for both T albacores and T obesus. The polymorphism of DNA profiles obtained by restriction digestion was used to differentiate the T albacores and T obesus species. Therefore, the current study carries a reliable approach to identify and distinguish T obesus and T albacores from the other tuna species. Keywords: Tuna species, DNA extraction, Polymerase chain reaction, Restriction Enzyme digestion
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    Expression of a Rabies Virus Specific Antigen by Cloning the Glycoprotein Gene into Escherichia con Expression System
    (Uva Wellassa University of Sri Lanka, 2016) Sewwandi, H.S.; Rodrigo, W.W.P.; Athapaththu, A.M.M.H.; Gunathilaka, P.A.D.H.N.; Bandara, K.G.W.W.; Wijesundara, R.R.M.K.K.; Bulumulla, P.B.A.I.K.
    Rabies is an infectious disease characterized by dysfunction of the central nervous system caused by Lyssavirus of family Rhabdoviridae. Detection of rabies antibodies are used to confirm if people have been successfully immunized. Currently, these detection methods require lots of expertise and are generally carried out in reference laboratories at a high cost. Therefore, it is vital to develop and standardize simple techniques such as Enzyme Linked Immunosorbent Assay (ELISA) for determining the level of antibodies against rabies virus at a lower cost. Hence, the aim of the present study was to clone rabies virus specific glycoprotein gene into bacterial expression vector for the production of recombinant protein. Initial attempts were made to isolate plasmid DNA of pET-28a (+) vector and pcDNA3. -RVG recombinant plasmid containing previously cloned Rabies Virus Glycoprotein gene (RVG). Both plasmids were successfully digested with BamHI and XhoI restriction enzymes. The purified Rabies Virus Glycoprotein gene was cloned into pET-28a (+) bacterial expression vector. The pET-28a (+)-RVG plasmids were successfully transformed into TOPIOP competent cells through electroporation. Transformants were screened by rapid screening method. Out of 20 colonies 8 were identified as recombinants. Further screening of recombinant colonies will be carried out by digesting with restriction enzymes. Putative correct recombinant construct will be transferred into bacterial expression system for the expression. Keywords: Rabies, Rabies virus glycoprotein gene, Cloning
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    Optimization of a Ribonucleic Acid (RNA) Extraction Protocol for Viruses in Clinical Samples for Disease Diagnosis
    (Uva Wellassa University of Sri Lanka, 2016) Kumar, S.A.; Gunathilaka, P.A.D.H.N.; Rodrigo, W.W.P.; Athapaththu, A.M.M.H.
    Ribonucleic acid (RNA) is a polymeric molecule. It is implicated in coding and gene expression. Some medically important organisms such as viruses have only RNAs as their inherent material. To detect the viral diseases using molecular biological methods, it needs extraction of RNA from body fluids. There are several methods of RNA extraction, which require costly reagents and kits. Hence, the objective of this study was to optimize a low cost, in-house protocol for RNA extraction of viruses in clinical samples in order to facilitate disease diagnosis. Clinically confirmed blood samples, which were positive for Dengue Virus by NS1 antigen test, were taken for optimization of the two protocols. Two different RNA extraction protocols were used for the study to identify the most appropriate and reliable method with high efficiency. Trizol reagent, which was prepared in house was used in both protocols. Extracted RNA from both the protocols were quantified at 260 nm using a spectrophotometer. The RNA amount quantified from the spectrophotometer showed a result of 64 and 72 ng/ul from first and second protocols, respectively. In the first protocol, all the procedures were undertaken at room temperature (27-35 °C) but generally RNA is not stable at the room temperature. Therefore, RNA might have degraded due to lack of optimum conditions during the incubation, centrifugation and storage periods. In addition, if the RNA pellets were air dried completely, it becomes insoluble in RNase free water. Therefore, extracted RNA might not have been re-suspended completely in the solution. Those identified drawbacks were adjusted in the second protocol. Further, incubation temperature and time period (4 °C and 30 minutes) and centrifugation time (15 minutes), were modified to achieve stabilization, complete precipitation of RNA molecules and to prevent degradation by RNases. According to the above discussed facts, this study reveals that the second protocol is more suitable for RNA extraction ofviruses in clinical samples. Keywords: Ribonucleic Acid (RNA), Virus, Extraction
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    Role of microorganisms against hydrocarbon contamination; Bioremediation
    (Uva Wellassa University of Sri Lanka, 2015) Arachchi, S.M.W.; Munasinghe, M.M.E.; Sabaragamukorale, S.T.; Abeygunaratne, S.S.; Rodrigo, W.W.P.; De Silva, D.P.D.C.; Dalpatadu, K.S.L.; Senaratne, S.G.; Gunathilaka, P.A.D.H.N.
    The development of human civilization has changed its path since the industrial revolution. Since then began the use of hydrocarbon sources as the primary energy source of the world. The use of oil as fuel has led to intensive economic development worldwide. Even though these compounds contribute to the global economy on massive scale they in turn have perilous effects on the biotic and abiotic components of the ecosystem. In the stages of oil refinement, transportation, storage and on daily activities, unavoidable oil spills take place in small amounts. However, the accidental large oil spills draw the attention of the public to find remediation solutions. The methods of remediation can be physical, chemical or biological or may be a combination of two or more of these techniques. Hydrocarbon utilizing bacteria, fungi and cyanobacteria have been found in soil, marine and fresh water ecosystems (Okoh, 2002). Although several countries have already used methods including microorganisms for bioremediation of petroleum spills, it has not been previously used in Sri Lanka. Therefore, the objective was to isolate indigenous bacterial strains from hydrocarbons contaminated soils to assess their potential for bioremediation and to develop a bio-product for bioremediation. Methodology Three sites with soil contaminated by different petroleum hydrocarbons were identified in Ceylon Petroleum Corporation, Sapugaskanda, Kelaniya, Sri Lanka. A total of 18 soil samples (6 from each site) were collected randomly by simple soil sampling method (American Society for Testing and Materials, 1998). A weight of 10 g of soil was diluted in 90 ml of 0.1% sterile Sodium pyrophosphate solution containing 30 g of sterile glass beads. After shaking the mixture for 1 hour at 175 rpm, the and were vortexed for 1 minute. A volume of 120 µl of each dilution was spread on Luria Broth (LB) agar medium and was incubated at 28 °C for 7 days. The colonies appeared were inoculated on a Bushnell Haas (BH) liquid and solid mediums supplemented with 50 µl of hydrocarbons followed by an incubation at 28 °C for 7 days. The identified colonies were subjected to genomic DNA extraction using the Phenol-Chloroform method. The extracted genomic DNA samples were sent over to Macrogen, Korea for 16S rRNA sequencing. The overnight grown bacterial cultures were centrifuged at 16000 g for 3 minutes at 4ºC. The pellet was resuspended in 200 µl of TE buffer and was vortexed and centrifuged at 16000 g for 1 minute at 4ºC and a volume of 1.5 µl of Protinase K was added and mixed. To this 20 µl (1/10) of 10% SDS was added, mixed well and incubated for 1 hour at 37 ºC. After the incubation, equal volume of Phenol: Chloroform (1:1) was added and centrifuged at 16000 g for 2 minutes at 4ºC. The aqueous layer was taken out without disturbing the protein layer and transferred into a fresh tube. A volume of 2V of 100% ice cold Ethanol and 0.1V Sodium acetate were added, mixed well and were incubated at 0 ºC for 1 hour. The solution mixture was centrifuged at 16000 g for 5 minutes at 4ºC. The supernatant was discarded and the pellet was dried and dissolved in 40 µl of nuclease-free water by tapping. For the selection of immobilizing agent, 10 g of autoclaved saw dust and rice husk each were mixed with 7.5 ml of Yeast Extract Glucose (YEG) broth separately and was autoclaved. Then the washed, pure bacterial cells were inoculated on to autoclaved rice husk and saw dust at room temperature and were incubated at 30 °C at 150 rpm for 5-6 days in a shaking incubator. The immobilized samples were washed with sterile saline water for 3 times and were inoculated on BH agar plates with diesel. Pure cultures of selected bacterial strains were inoculated with LB agar and were incubated over- night. A single colony of each bacterial strain was inoculated on 5 ml of LB broth. The cultured cells were centrifuged at 2000 g at 4°C for 10 minutes and the pellet was dissolved in 5 mL of phosphate buffer and re-centrifuged under the same conditions. Then the pellet was re-suspended in 5 ml of phosphate buffer. A mass of 14 g of autoclaved rice husk were mixed with 21 ml of YEG broth and was autoclaved. Then 2 ml of washed Bacterial cultures were inoculated on 2 g of autoclaved rice husk at room temperature separately and were incubated at 30 °C at 150 rpm for 5-6 days in a shaking incubator until a heavy culture develops. A volume of 20 ml of water was contaminated with 2 ml of diesel and 0.2 g of immobilized rice husk was added on top of the oil layers under sterile conditions. Turbidity and the time taken for the disruption of oil layer in the water were compared with a control.